Fetal alcohol spectrum disorder is common (affects ~2% of all live births) and is a major cause of mental dysfunction. Of the many negative effects that ethanol has on the developing nervous system, the most severe is neuronal death. It is severe because this loss is permanent; with the exception of a couple of sites, post-mitotic neurons in the CNS are not replaced by newly generated ones. Indeed, after the generation of neurons is complete, the nervous system has to decide if and whether damaged neurons can be repaired. If not, they must be eliminated. A key molecule in this decision process is the oncoprotein p53. p53 is critical for maintaining neuronal integrity and resiliency. We will test the hypothesis that developmental exposure to ethanol alters neuronal survival and DNA repair through p53-dependent activities. In the developing nervous system, neuronal death is a natural and critical process. This death appears to be apoptotic and to involve p53. Our preliminary data and the work of others on developing cerebral cortex show that exposure to ethanol during the period of naturally occurring neuronal death causes a dramatic and transient increase in both active caspase 3 expression and terminal uridylated nick-end labeling (TUNEL). On the other hand, our novel data also show that this pattern does not coincide with the ultimate loss of cortical neurons either in time or space. The implication is that ethanol causes DNA fragmentation, but this degradation may not be obligatory for apoptosis. Instead, it may reveal DNA repair mechanisms. p53 is a key player in DNA repair. Three complementary aims will be addressed using p53 deficient mice and cells. (1) Vulnerability of cortical neurons to ethanol will be addressed in long- and short-term in vivo studies. Long-term studies will determine the ethanol-induced loss of neurons in cortical layers occurring in the deficient mice. Complementary studies will examine short-term changes in the expression of presumed death markers (active caspase3 immunoexpression and TUNEL) in cortical layers and the timing of the neuronal loss. (2) The acute genomic responses of p53 deficient mice and cultured neural stem cells to ethanol will be determined. We will identify changes in the expression of transcripts involved in apoptosis and DNA repair. In addition, we will determine the epigenetic effects of ethanol on the silencing of genes through hyper-methylation. (3) Securin is a protein that is regulated by p53 and is pivotal for DNA repair. Preliminary microarray and immunocytochemical data show that it is profoundly affected by ethanol. Thus, we will examine the effects of ethanol on cells deficient of securin in vitro and in vivo after transplantation into layers that are apparently susceptible and refractory to ethanol. These studies will explore two dovetailed responses that developing neurons have to ethanol: DNA repair, and failing that, apoptotic death. We will address critical questions. For example, what defines the susceptibility of a young neuron to ethanol? Can neurons be manipulated to reduce their ethanol vulnerability? Thus, the studies explore two new targets of ethanol, p53 and securin, that are critical for the neuronal survival and integrity.